Cyanocobalamin low viscosity aqueous formulations for intranasal delivery

ABSTRACT

A stable pharmaceutical mercury-free aqueous solution of cyanocobalamin comprised of cyanocobalamin and water wherein said solution of cyanocobalamin is suitable for intranasal administration, has a viscosity less than about 1000 cPs, and wherein said solution of cyanocobalamin has a bioavailability of cyanocobalamin when administered intranasally of at least about 7% relative to an intramuscular injection of cyanocobalamin with the proviso that the solution is essentially free of mercury and mercury-containing compounds. The present invention is also directed towards a method for elevating the vitamin B12 levels in the cerebral spinal fluid (CSF) comprising administering intranasally a sufficient amount of a mercury-free cyanocobalamin solution so as to increase the average ratio of vitamin B12 in the CSF to that in the blood serum (B12 CSF/B12 Serum×100) to at least about 1.1 comprising intranasally administering an aqueous solution of a cyanocobalamin, wherein said solution of cyanocobalamin has a bioavailability of at least 7% relative to an intramuscular injection of a cyanocobalamin.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division and claims priority under Title 35, U.S.Code, § 120 of co-pending U.S. patent application Ser. No. 10/814,399filed on Mar. 31, 2004, currently allowed, which is acontinuation-in-part and claims priority under Title 35, U.S. Code, §120 of U.S. patent application Ser. No. 10/787,385 filed on Feb. 26,2004, now U.S. Pat. No. 7,229,636 issued Jun. 12, 2007, which claims thebenefit under 35 U.S.C. §119(e) of U.S. Provisional Application No.60/451,899 filed on Mar. 4, 2003, U.S. Provisional Application No.60/461,583 filed on Apr. 8, 2003, and U.S. Provisional Application No.60/474,204 filed on May 29, 2003, each of which is incorporated byreference herein in its entirety.

BACKGROUND OF THE INVENTION

Vitamin B12 is a dietary essential, a deficiency of which results indefective synthesis of DNA in any cell in which chromosomal replicationand division are taking place. Since tissues with the greatest rate ofcell turnover show the most dramatic changes, the hematopoietic systemis especially sensitive to vitamin B12 deficiencies. An early sign ofB12 deficiency is a megaloblastic anemia. Dietary B12, in the presenceof gastric acid and pancreatic proteases, is released from food andsalivary binding protein and bound to gastric intrinsic factor. When thevitamin B12-intrinsic factor complex reaches the ileum, it interactswith a receptor on the mucosal cell surface and is actively transportedinto circulation. Adequate intrinsic factor, bile and sodium bicarbonate(suitable pH) all are required for ileal transport of vitamin B12.Vitamin B12 deficiency in adults is rarely the result of a deficientdiet; rather, it usually reflects a defect in one or another aspect ofthis complex sequence of absorption. Achlorhydria and decreasedsecretion of intrinsic factor by parietal cells secondary to gastricatrophy or gastric surgery is a common cause of vitamin B12 deficiencyin adults. Antibodies to parietal cells or intrinsic factor complex alsocan play a prominent role in producing deficiency. A number ofintestinal diseases can interfere with absorption. Vitamin B12malabsorption is seen with pancreatic disorders (loss of pancreaticprotease secretion), bacterial overgrowth, intestinal parasites, sprue,and localized damage to ileal mucosal cells by disease or as a result ofsurgery. The recommended daily intake of vitamin B12 in adults is 2.4μg.

There are four main forms of vitamin B12: cyanocobalamin:hydroxocobalamin, methylcobalamin and adenosylcobalamin. Methylcobalaminand adenosylcobalamin are unstable and damaged by light. They aretherefore unsuitable for use in dietary supplements or pharmaceuticalsand are not essential since they can be formed from cyanocobalamin orhydroxocobalamin within the body. The main form of vitamin B12 found infood is hydroxocobalamin. The main form used therapeutically and innutritional supplements is cyanocobalamin, chosen because it is the moststable form and therefore easiest to synthesize and formulate.

Because deficiencies of vitamin B12 are generally caused by theinability of the vitamin to be absorbed in the small intestine due to abreakdown in the vitamin B12-intrinsic factor complex transportmechanism, vitamin B12 must therefore be administered systemically.Currently, therapeutic amounts of cyanocobalamin are administered byintramuscular or deep subcutaneous injection of cyanocobalamin. However;patients must return to the physician's office periodically to receiveadditional injections to maintain their levels of vitamin B12. However,an intranasal gel cyanocobalamin preparation, NASCOBAL® is currentlybeing marketed in which cyanocobalamin is administered intranasally asmaintenance vitamin B12 therapy. However, many patients find theconsistency of the intranasal gel unpleasant and would prefer to haveadministered intranasally a low viscosity spray containingcyanocobalamin.

The prior art suggests that for vitamin B12 to be absorbed intranasallyin therapeutically beneficial amounts, the concentration of the B12 insolution must either be greater that 1% by weight, see Merkus, U.S. Pat.No. 5,801,161 or be administered intranasally in a viscous gel, Wenig,U.S. Pat. No. 4,724,231 so that the gel remains in the nostril for anextended period of time. In fact Wenig states that B12 administeredintranasally in a low viscosity solution is not in contact with thenasal mucosa long enough for a sufficient period of time to permituseful absorption. Wenig claims that most of the B12 is wasted if thesolution has a low viscosity. Merkus developed intranasal formulationsof hydroxocobalamin having a concentration of hydroxocobalamin greaterthan 1%, however hydroxocobalamin is not very stable and thus has ashort shelve-life. Merkus chose hydroxocobalamin because cyanocobalaminis not soluble in an aqueous solution at concentrations greater than 1%.

U.S. Pat. No. 4,525,341, Deihl, discloses a method of administeringvitamins intranasally but do not enable a specific formulationcontaining only cyanocobalamin. International Patent Application No.PCT/US86/00665, publication no. WO 86/05987, discloses nasal spraycomposition containing vitamin B12 as cyanocobalamin. However, thespecific spray formulations all contained a mercury compound as apreservative, however the disclosure did require the presence of mercurycompounds. Other preservatives were also mentioned includingbenzalkonium chloride and chlorobutanol. As was stated above, anintranasal gel containing cyanocobalamin, NASCOBAL®, is currently beingproduced and marketed by Nastech Pharmaceutical Company Inc. of Bothell,Wash. It is very effective in maintaining levels of vitamin B12 forpatients who have been deficient in the past but have recovered theirlevels of B12 through intramuscular injections. However, a number ofpatients find the consistency of the gel unpleasant in their nose, andwould prefer an intranasal formulation that has a lower viscosity and isfree of mercury compounds. Thus, there is a need to produce apharmaceutically stable aqueous solution of cyanocobalamin that has alow viscosity, is free of mercury compounds and has sufficientbioavailability to be used as a maintenance therapy for vitamin B12.

SUMMARY OF THE INVENTION

The present invention fills this need by providing for a stablepharmaceutical solution of cyanocobalamin suitable for intranasaladministration, having a viscosity less than about 1000 cPs, whereinsaid intranasal solution of cyanocobalamin has a bioavailability of atleast 7% of the bioavailability of an intramuscular injection ofcyanocobalamin and is free of mercury compounds.

A preferred formulation is comprised of cyanocobalamin, citric acid,sodium citrate, and water wherein the viscosity is less than 1000 cPs,and wherein the solution of cyanocobalamin has a bioavailability of atleast 8%, more preferably at least about 9, 10, 11, or 12% of thebioavailability of an intramuscular injection of cyanocobalamin.

Preferred compositions within the scope of this invention will contain ahumectant to inhibit drying of the mucous membranes and to preventirritation. Any of a variety of humectants can be used including but notlimited to sorbitol, propylene glycol or glycerol. A preferred humectantis glycerin.

A preservative is generally employed to increase the shelf life of thecompositions. Examples of preservative include but are not limited tobenzyl alcohol, chlorobutanol and benzalkonium chloride. A preferredpreservative is benzalkonium chloride. A suitable concentration of thepreservative will be from 0.002% to 2.0% based upon the total weight,although there may be appreciable variation depending upon the agentselected.

A most preferred formulation has the concentration of cyanocobalamin at0.5% (percent of total weight), citric acid 0.12%, sodium citrate 0.32%,glycerin 2.23%, benzalkonium chloride 0.02% and 96.79% water.

Another embodiment of the present invention is a method foradministering cyanocobalamin comprised of infusing the nose with anaqueous solution of cyanocobalamin, wherein the solution ofcyanocobalamin has a viscosity of less than 1000 cPs, and wherein saidsolution of cyanocobalamin has a bioavailability of at least about 7%relative to an intramuscular injection of cyanocobalamin. Preferably,the bioavailability is at least 8%, 9%, 11% or 12%.

The present invention is further directed towards a method for elevatingthe vitamin B12 levels in the cerebral spinal fluid (CSF) comprisingintranasally administering a solution of cyanocobalamin so as toincrease the average ratio of vitamin B12 in the CSF to that in theblood serum (B12 CSF/B12 Serum×100) to at least about 1.1, wherein saidsolution of cyanocobalamin has a bioavailability of at least 7% relativeto an intramuscular injection of cyanocobalamin. In a more preferredembodiment the B12 CSF levels are increased so that the ratio of B12 inthe CSF to the levels in the blood serum is at least 1.9.

The methods of present invention are further comprised of thecyanocobalamin solutions being administered into a nose of an individualthrough an actuator tip as a spray, wherein the spray preferably has oneor more of the following properties: a spray pattern ellipticity ratioof from about 1.0 to about 1.4 when measured at a height of 3.0 cm fromthe actuator tip; or the spray produces droplets, less than about 5% ofthe droplets are less than 10 μm in size; the spray has a spray patternmajor axis of about 35.3 mm and a minor axis of about 30.8 mm; 50% ofthe droplets produced by the spray are 26.9 μm or less in size; 90% ofthe droplets produced by the spray are 55.3 μm or less in size; or 10%of the droplets produced by the spray are 12.5 μm or less in size.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a nasal spray pump kit containing the cyanocobalaminsolution of the present invention having an actuator that is notengaged.

FIG. 1B shows the nasal spray pump kit containing the cyanocobalaminsolution of the present invention having an actuator that is engaged andexpelling a spray plume of the cyanocobalamin solution of the presentinvention.

FIG. 2 shows the spray pattern produced by the actuator of the spraypump kit.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions may aid in the understanding of the presentinvention.

“About”: is taken to be a relative term denoting an approximation ofplus or minus 20% of the nominal value it refers to. For the field ofpharmacology and clinical medicine and analogous arts that are thesubject of this disclosure, this level of approximation is appropriateunless the value is specifically stated to be critical or to require atighter range.

“Nasal mucosa”: the nasal mucosa is taken to be the lining of thevestibule of the nose, where vascularized, and extending interiorly tothe boundaries of the oropharynx and sinuses.

“Aqueous”: refers to a solution formed in water, but may contain lesseramounts of other co-solvents.

“Bioavailability” is defined as the rate and extent to which the activeingredient or active moiety is absorbed from a drug product and becomesavailable at the site of action, [21 CFR §320.1(a)].

“Bioavailability of the intranasal spray relative to an intramuscularinjection of cyanocobalamin” means the percent amount a dose of theintranasal taken up by the systemic vascular system in comparison to thesame amount of cyanocobalamin injected. For example, assuming anintramuscular injection of a solution of cyanocobalamin containing 100μg of cyanocobalamin would have a 100% bioavailability, if an intranasaldose of cyanocobalamin contains 100 μg and has at least 7%bioavailability relative to an injection of cyanocobalamin, at least 7μg of cyanocobalamin would be taken up into the blood vasculature.Likewise, if the intranasal dose of cyanocobalamin contained 500 μg, atleast 35 μg of cyanocobalamin would be taken up into the bloodvasculature, if the intranasal formulation had a bioavailability of atleast 7%.

“Stability”: during storage, any compositional change measured in aparameter, examples of which include but are not limited toconcentration, degradation, viscosity, pH, or particle size, that isconsidered to significantly affect the quality attributes of the productover time, denotes instability. In a similar vein, changes that are notconsidered to significantly affect the quality attributes of the productconnote stability. The time period over which stability is measured isrelative depending on the intended utility of the composition.Accelerated stability at higher temperature is sometimes taken as a morespeedy way of extrapolating stability over longer periods of time thanare actually measured.

“Pharmaceutically acceptable”: refers to a composition which whenadministered to a human or a mammal by the indicated route ofadministration, provokes no adverse reaction which is disproportionateto the benefit gained by administration of said compound.

“Mammal” shall include any of a class of warm-blooded higher vertebratesthat nourish their young with milk secreted by mammary glands and haveskin usually more or less covered with hair, and non-exclusivelyincludes humans and non-human primates, their children, includingneonates and adolescents, both male and female, livestock species, suchas horses, cattle, sheep, and goats, and research and domestic species,including dogs, cats, mice, rats, guinea pigs, and rabbits. “Patient” or“subject” is used herein interchangeably with “mammal.”

“Intranasal delivery” shall mean delivery of a drug primarily via themucosa of the nasal cavity. This includes the superior, middle andinferior nasal turbinates and the nasal pharynx. Note that the olfactoryregion is concentrated in the superior (upper ⅓) of the nasalturbinates. Cilial action pushes material back toward the oropharynx, somaterial deposited in the nasal vestibule encounters the nasal mucosabefore entering the throat.

“Substantially free” refers to the level of a particular activeingredient in the compositions of the invention, wherein the particularactive ingredient constitutes less than 20%, preferably less than 10%,more preferably less than 5%, and most preferably less than 1%, byweight based on the total weight of active ingredients in thecomposition.

Delivery vehicles herein found useful include actuator dispenserscommonly used for nasal solutions and gels. Embodiments of thistechnology include multiple, single-dose, metered dose, child resistant,and disposable dispensers, and their kits.

As used herein “peak concentration (C_(max)) of cyanocobalamin in ablood plasma”, “area under concentration vs. time curve (AUC) ofcyanocobalamin in a blood plasma”, “time to maximal plasma concentration(t_(max)) of vitamin in a blood plasma” are pharmacokinetic parametersknown to one skilled in the art. [Laursen et al., Eur. J. Endocrinology,135: 309-315, (1996)]. The “concentration vs. time curve” measures theconcentration of cyanocobalamin in a blood serum of a subject vs. timeafter administration of a dosage of cyanocobalamin to the subject eitherby intranasal, subcutaneous, or other parenteral route ofadministration. “C_(max)” is the maximum concentration of cyanocobalaminin the blood serum of a subject following a single dosage ofcyanocobalamin to the subject. The term “t_(max)” is the time to reachmaximum concentration of cyanocobalamin in a blood serum of a subjectfollowing administration of a single dosage of cyanocobalamin to thesubject.

As used herein, “area under concentration vs. time curve (AUC) ofcyanocobalamin in a blood plasma” is calculated according to the lineartrapezoidal rule and with addition of the residual areas. A decrease of23% or an increase of 30% between two dosages would be detected with aprobability of 90% (type II error β=10%). The “delivery rate” or “rateof absorption” is estimated by comparison of the time (t_(max)) to reachthe maximum concentration (C_(max)). Both C_(max) and t_(max) areanalyzed using non-parametric methods. Comparisons of thepharmacokinetics of subcutaneous, intravenous and intranasalcyanocobalamin administrations were performed by analysis of variance(ANOVA). For pair wise comparisons a Bonferroni-Holmes sequentialprocedure was used to evaluate significance. The dose-responserelationship between the three nasal doses was estimated by regressionanalysis. P<0.05 was considered significant. Results are given as meanvalues +/−SEM. (Laursen et al., 1996.)

The above-described cyanocobalamin solutions are designed to beadministered to the nasal mucosa either in drop or in spray form.However, the preferred mode of administration is in spray form, i.e., inthe form of finely divided droplets. An example of a suitable spray pumpis the Pfeiffer Spray Pump Model # 63385 produced by Pfeiffer GmbH,Radolfzell, Germany.

Nasal Administration of Cyanocobalamin

Cyanocobalamin is administered intranasally using a nasal sprayaccording to the present invention. In this area the followingdefinitions are useful.

-   -   1. Aerosol—A product that is packaged under pressure and        contains therapeutically active ingredients that are released        upon activation of an appropriate valve system.    -   2. Metered aerosol—A pressurized dosage form comprised of        metered dose valves, which allow for the delivery of a uniform        quantity of spray upon each activation.    -   3. Powder aerosol—A product that is packaged under pressure and        contains therapeutically active ingredients in the form of a        powder, which are released upon activation of an appropriate        valve system.    -   4. Spray aerosol—An aerosol product that utilizes a compressed        gas as the propellant to provide the force necessary to expel        the product as a wet spray; it generally applicable to solutions        of medicinal agents in aqueous solvents.    -   5. Spray—A liquid minutely divided as by a jet of air or steam.    -   6. Metered spray—A non-pressurized dosage form consisting of        valves that allow the dispensing of a specified quantity of        spray upon each activation.    -   7. Suspension spray—A liquid preparation containing solid        particles dispersed in a liquid vehicle and in the form of        course droplets or as finely divided solids.

The fluid dynamic characterization of the aerosol spray emitted bymetered nasal spray pumps as a drug delivery device (“DDD”). Spraycharacterization is an integral part of the regulatory submissionsnecessary for Food and Drug Administration (“FDA”) approval of researchand development, quality assurance and stability testing procedures fornew and existing nasal spray pumps.

Thorough characterization of the spray's geometry has been found to bethe best indicator of the overall performance of nasal spray pumps. Inparticular, measurements of the spray's divergence angle (plumegeometry) as it exits the device; the spray's cross-sectionalellipticity, uniformity and particle/droplet distribution (spraypattern); and the time evolution of the developing spray have been foundto be the most representative performance quantities in thecharacterization of a nasal spray pump. During quality assurance andstability testing, plume geometry and spray pattern measurements are keyidentifiers for verifying consistency and conformity with the approveddata criteria for the nasal spray pumps.

DEFINITIONS

Plume Height—the measurement from the actuator tip to the point at whichthe plume angle becomes non-linear because of the breakdown of linearflow. Based on a visual examination of digital images, and to establisha measurement point for width that is consistent with the farthestmeasurement point of spray pattern, a height of 30 mm is defined forthis study

Major Axis—the largest chord that can be drawn within the fitted spraypattern that crosses the COMw in base units (mm)

Minor Axis—the smallest chord that can be drawn within the fitted spraypattern that crosses the COMw in base units (mm)

Ellipticity Ratio—the ratio of the major axis to the minor axis

D₁₀—the diameter of droplet for which 10% of the total liquid volume ofsample consists of droplets of a smaller diameter (μm)

D₅₀—the diameter of droplet for which 50% of the total liquid volume ofsample consists of droplets of a smaller diameter (μm), also known asthe mass median diameter

D₉₀—the diameter of droplet for which 90% of the total liquid volume ofsample consists of droplets of a smaller diameter (μm)

Span—measurement of the width of the distribution. The smaller thevalue, the narrower the distribution. Span is calculated as

$\frac{\left( {D_{90}\text{-}D_{10}} \right)}{D_{50}}$

% RSD—relative standard deviation, the standard deviation divided by themean of the series and multiplied by 100, also known as % CV.

Cyanocobalamin Nasal Spray Kit.

The present invention is further comprised of a cyanocobalamin nasalspray kit and method of administering the cyanocobalamin solution usingthe nasal spray kit.

The nasal spray kit is exemplified by FIGS. 1A, 1B and FIG. 2. FIGS. 1Aand 1B show a nasal spray device 10 before engagement (FIG. 1A) andafter engagement (FIG. 1B). The cyanocobalamin nasal spray kit, 10, iscomprised of a container, in this case a bottle 12 into which thecyanocobalamin formulation is placed, and an actuator 14, attached tobottle 12 and in fluid connection with the solution of cyanocobalamin inbottle 12. When the actuator, 14 is actuated or engaged, it forces aspray plume, 16 of cyanocobalamin through tip 15 of the actuator. Thespray plume is comprised of droplets of the solution of cyanocobalamin.A spray pattern is determined by taking a photograph of a cross-sectionof the spray plume 16 above a predetermined height, of the plume. Thespray plume also has angle of ejection, 20, as it leaves actuator, 14. Aspray pattern of spray plume 16 is shown on FIG. 2. The Spray pattern ofFIG. 2, is elliptical and has a major axis, 24, and a minor axis 26.

In a preferred embodiment, the actuator produces spray of thecyanocobalamin solution having a spray pattern ellipticity ratio of fromabout 1.0 to about 1.4 when measured at a height of 3.0 cm from theactuator tip. In a preferred embodiment less than 5% of the droplets ofthe cyanocobalamin solution are less than 10 μm in size, the spraypattern has a major axis and minor axis of 25 and 40 mm, respectively,50% of the droplets are 26.9 μm or less in size, 90% of the droplets are55.3 μm or less in size, and 10% of the droplets are 12.5 μm or less insize.

As noted above, the present invention provides improved methods andcompositions for intranasal delivery cyanocobalamin to mammaliansubjects for treatment or prevention of a variety of diseases, disordersand conditions. Examples of appropriate mammalian subjects for treatmentand prophylaxis according to the methods of the invention include, butare not restricted to, humans and non-human primates, livestock species,such as horses, cattle, sheep, and goats, and research and domesticspecies, including dogs, cats, mice, rats, guinea pigs, and rabbits.

An initial therapy, the patient should receive daily intramuscularinjections of 100 μg of cyanocobalamin for about 1 to 2 weeks, togetherwith 1 to 5 mg of folic acid. Intramuscular injections of cyanocobalaminshould not be greater than 100 μg as doses in excess of 100 μg arerapidly cleared from the plasma into the urine, and administration oflarger amounts of vitamin B12 will not result in greater retention oflarger amounts of the vitamin.

The cyanocobalamin nasal spray of the present invention is directedtowards the maintenance of the hematological status of patients who arein remission following intramuscular vitamin B12 therapy. So instead ofa once a month injection of 100 μg of cyanocobalamin, using thecyanocobalamin spray, the patient self-administers a dose of the nasalspray of the present invention containing 500 μg of cyanocobalamin onceor twice a week. The maintenance therapy of the intranasalcyanocobalamin is for any patient that had been diagnosed with a vitaminB12 deficiency, but especially for those treated for pernicious anemiaand dietary deficiency of vitamin B12 occurring in strict vegetarians,the so-called vegans who eat no animal products. Maintenancecyanocobalamin therapy using the cyanocobalamin solution of the presentinvention is also indicated for those afflicted with malabsorption ofvitamin B12 resulting from structural or functional damage to thestomach, where intrinsic factor is secreted or to the ileum, whereintrinsic factor facilitates B12 absorption. These conditions includetropical sprue and nontropical sprue (Idiopathic steatorrhea,gluten-induced enteropathy).

Maintenance cyanocobalamin therapy using the cyanocobalamin solution ofthe present invention is also indicated for those afflicted withmalabsorption of vitamin B 12 resulting from inadequate secretion ofintrinsic factor, resulting from lesion that destroys the gastric mucosa(ingestion of corrosives, extensive neoplasia), and a number ofconditions associated with a variable degree of gastric atrophy (such asmultiple sclerosis, human immunodeficiency viral (HIV) infection certainendocrine disorders, iron deficiency, and subtotal gastrectomy).Structural lesions that lead to B12 deficiency include ileitis, ilealresections, Crohn's disease and malignancies. Vitamin B12 deficienciesmay also be the result of competition by intestinal parasites, andinadequate utilization of vitamin B12 occurring if antimetabolites forthe vitamin are employed in the treatment of neoplasia.

The intranasal cyanocobalamin solution of the present invention can alsobe used for individual who require above normal levels of vitamin B12,due to for example pregnancy, thyrotoxicosis, hemolytic anemia,hemorrhage, malignancy, hepatic and renal disease.

As was stated above, the present invention provides for a stablepharmaceutical solution of cyanocobalamin suitable for intranasaladministration, having a viscosity less than about 1000 cPs, whereinsaid intranasal solution of cyanocobalamin has when administeredintranasally a bioavailability of at least 7% of the bioavailability ofan intramuscular injection of cyanocobalamin. The intranasal formulationwill generally be comprised of in addition to water and cyanocobalamin,a buffering agent to maintain the pH between 4 and 6 preferably about 5,a humectanct to inhibit drying of the mucous membranes and apreservative.

A preferred formulation is comprised of cyanocobalamin, citric acid,sodium citrate, and water wherein the viscosity is less than 1000 cPs,and wherein the solution of cyanocobalamin has a bioavailability of atleast 7%, more preferably at least about 8, 9, 10, 11, 12% or more ofthe bioavailability of an intramuscular injection of cyanocobalamin.

Preferred compositions within the scope of this invention will contain ahumectant to inhibit drying of the mucous membranes and to preventirritation. Any of a variety of humectants can be used including, forexample sorbitol, propylene glycol or glycerol. A preferred humectant isglycerin.

A preservative is generally employed to increase the shelf life of thecompositions. Examples of preservative include benzyl alcohol, parabensthimerosal, chlorobutanol, benzethonium chloride and benzalkoniumchloride. A preferred preservative is benzalkonium chloride. A suitableconcentration of the preservative will be from 0.002% to 2% based uponthe total weight, although there may be appreciable variation dependingupon the agent selected.

A most preferred formulation has the concentration of cyanocobalamin at0.5% (percent of total weight), citric acid 0.12%, sodium citrate 0.32%,glycerin 2.23%, benzalkonium chloride solution 0.02% and 96.79% water.

Other buffering agent combination include but are not limited to:

Monopotassium phosphate and disodium phosphate,

Potassium biphthalate and sodium hydroxide, and Sodium acetate andacetic acid.

Another embodiment of the present invention is a method foradministering cyanocobalamin comprised of infusing the nose with anaqueous solution of cyanocobalamin, wherein the solution ofcyanocobalamin has a viscosity of less than 1000 cPs, and wherein saidsolution of cyanocobalamin has a bioavailability of at least 7% relativeto an intramuscular injection of cyanocobalamin. Preferably thebioavailability is at least about 8, 9, 10, 11, 12% or more of thebioavailability of an intramuscular injection of cyanocobalamin.

The present invention is further directed towards a method for elevatingthe vitamin B12 levels in the cerebral spinal fluid (CSF) comprisingintranasally administering a solution of cyanocobalamin so as toincrease the average ratio of vitamin B12 in the CSF to that in theblood serum (B12 CSF/B12 Serum×100) to at least about 1.1, wherein saidsolution of cyanocobalamin has a bioavailability of at least 7% relativeto an intramuscular injection of a cyanocobalamin. In a more preferredembodiment the B12 CSF levels are increased so that the ratio of B12 inthe CSF to the levels in the blood serum is at least 1.9.

This is a significant embodiment of the present invention becausevitamin B12 deficiency can result in irreversible damage to the nervoussystem. Progressive swelling of myelinated neurons, demyelination, andneuronal cell death are seen in the spinal column and cerebral cortex.This causes a wide range of neurological signs and symptoms, includingparesthesias of the hands and feet, diminution of vibration and positionsenses with resultant unsteadiness, decreased deep tendon reflexes, and,in the later stages, confusion, moodiness, loss of memory, and even aloss of central vision. The patient may exhibit delusions,hallucinations, or even an overt psychosis. Since the neurologicaldamage can be dissociated from the changes in the hematopoietic, vitaminB12 deficiency must be considered as a possibility in elderly patientswith dementia and psychiatric disorders, even if they are not anemic.Thus, the embodiment of the present invention directed towardsincreasing the level of vitamin B12 in the CSF can have tremendousbenefit for neurological patients. Thus, intranasal administration ofvitamin B12 can be used to treat such diseases as Alzheimer's disease,dementia, and multiple sclerosis.

Preferred formulations are the following: Cyanocobalmin Nasal Spray 500mcg/0.1 mL

Formulation:

Current Nasal Solution Component. Quantity (% w/w) Cyanocobalamin, USP0.50 Citric acid anhydrous, USP 0.12 Sodium citrate dihydrate, USP 0.32Glycerin, USP 2.23 Benzalkonium chloride (50%), 0.04 NF Purified waterq.s. 100.0

Alternative buffer systems and amounts that can be used forCyanocobalamin Nasal Spray

Quantity (% w/w) 1) Citric Acid-Phosphate buffer Citric Acid anhydrous,USP 0.240 Dibasic Sodium Phosphate anhydrous 0.357 2) Acetate bufferSodium Acetate anhydrous, USP 0.220 Acetic Acid, glacial, USP 0.064 3)Phosphate buffer Monobasic Potassium Phosphate anhydrous, NF 0.483Dibasic Sodium Phosphate anhydrous 0.004

The intranasal formulations of the present invention can be administeredusing any spray bottle or syringe. A preferred nasal spray bottle isthe, “Nasal Spray Pump w/Safety Clip, Pfeiffer SAP # 60548, whichdelivers a dose of 0.1 mL per squirt and has a diptube length of 36.05mm. It can be purchased from Pfeiffer of America of Princeton, N.J.

The following examples are provided by way of illustration, notlimitation.

EXAMPLE 1 Comparison of Intranasal Cyanocobalamin Solution of thePresent Invention with NASCOBAL® and Intramuscular Injections ofCyanocobalamin Introduction

Nascobal® (Cyanocobalamin, USP) is a synthetic form of vitamin B₁₂ withequivalent vitamin B₁₂ activity. The chemical name is 5,6-dimethyl-benzimidazolyl cyanocobamide. Currently, Nascobalo®(Cyanocobalamin, USP) is marketed as a self-administered nasal gel. Therecommended dose of Nascobal® (Cyanocobalamin, USP) in subjects withvitamin B₁₂ malabsorption who are in remission following inject ablevitamin B₁₂ therapy is 500-μg administered intranasally once weekly.

Vitamin B₁₂ deficiency has a number of causes, including malabsorptionof vitamin B₁₂ resulting from structural or functional damage to thegastrointestinal system and dietary deficiency of vitamin B₁₂.

The purposes of this study are to compare the bioequivalence of vitaminB₁₂ nasal gel versus the nasal spray, and to evaluate the relativebioavailability of three preparations of vitamin B₁₂ in a fasted statein normal healthy male and female subjects.

Intranasal cyanocobalamin gel is approved for a dose of 500 μg. Thecurrent study also utilizes a cyanocobalamin nasal spray at the same 500μg dose and an intramuscular dose of 100 μg.

Study Objectives

To compare the pharmacokinetic profile of a singleintranasally-administered spray, single intranasally-administered gel(Nascobal®), and single intramuscular-administered vitamin B₁₂ in afasted state in normal healthy male and female subjects.

Investigational Plan

Overall Study Design and Plan

This study was a single-site, open-label, 3-way (3-treatment,6-sequence) crossover, pharmacokinetic study of vitamin B12 administeredvia intranasal (IN) spray (500-μg), IN gel (Nascobal®) (500-μg), andintramuscular (IM) injection (100-μ) in fasted normal healthy male andfemale subjects, as follows:

Treatment A: One IN spray administration of 500-μg vitamin B₁₂. Theintranasal formulation was comprised of a preferred embodiment of thepresent invention and contained cyanocobalamin at a concentration of0.5% (percent of total weight), citric acid 0.12%, sodium citrate 0.32%,glycerin 2.23%, 50% benzalkonium chloride solution 0.04% and 96.79%water.

Treatment B: One IN gel administration of 500-μg vitamin B₁₂(Nascobal®).

Treatment C: One IM administration of 100-μg vitamin B₁₂.

Subjects were on a Vitamin B₁₂-free diet throughout each confinementperiod. Subsequent treatments will be dosed no sooner than 14 daysfollowing the preceding treatment dose administration.

Treatments Treatments Administered

On Day 1 of Periods I, II, and III after an 8 hour fast, subjectsreceived a single IN spray of 500-μg vitamin B₁₂ (Treatment A), a singleIN gel of 500-μg vitamin B₁₂ (Nascobal®) (Treatment B), or a single IMadministration of 100-μg vitamin B₁₂ (Treatment C) based upon arandomization generated by the PPD Development Biostatistician in one ofsix sequences. Following all periods, all subjects were to have receivedeach treatment in a crossover manner. A washout period of 14 daysseparated the three dosing periods.

On the morning of Day 1, subjects assigned to Treatment A received asingle IN spray administration of 500 μg of vitamin B₁₂. Subjectsassigned to Treatment B received a single IN gel administration of 500μg of vitamin B₁₂ (Nascobal®). Subjects assigned to Treatment C receiveda single IM administration of 100 μg of vitamin B₁₂. Doses were precededby an overnight fast (i.e., at least 8 hours) from food (not includingwater) and were followed by a fast from food (not including water) forat least 4 hours post-dose.

While confined at the clinical site, subjects received a standardizedvitamin B₁₂-deficient diet at scheduled times which did not conflictwith other study-related activities. A registered dietician set up thediet, and the food staff maintained a diet diary. No dietary supplementswere permitted during the study. Subjects abstained from consumingalcohol-containing, grapefruit-containing, or caffeine-containing foodsor beverages for 72 hours prior to Check-in.

Study Variables

For each subject, the following pharmacokinetic parameters werecalculated whenever possible, based on the serum concentrations ofvitamin B₁₂ from Treatments A, B, and C according to the modelindependent approach: C_(max), T_(max), and AUC_(0-t)

Pharmacokinetic Measurements

Blood samples for PK analysis of vitamin B₁₂ levels were collected viaan indwelling catheter and/or via direct venipuncture using 5-mLyellow-top Vacutainer® Hemogard™ evacuated serum separator collectiontube. Blood samples for PK analysis of vitamin B₁₂ levels were collectedon Day −1 at 0, 6, and 12 hours and Day 1 at 0 hour (i.e., pre-dose); 30minutes; 1, 1.5, 2, 4, 6, 8, 10, 12, 18, 24, 36, 48, 60, 72, 84 and 96hours post-dose during each period.

Appropriateness of Measurements

The pharmacokinetic parameters used in this study were those typicallyused to assess bioequivalence. All assessments of bioequivalence werebased on comparisons of AUC_(0-t), T_(max), and C_(max) (test versusreference treatments).

AUC is a measure of the extent of drug bioavailability and reflects thetotal amount of drug that reaches the systemic circulation.

C_(max) represents the maximum serum concentration obtained after drugadministration and provides an indication that sufficient drug hasreached the systemic circulation to provide a therapeutic response. Inaddition, C_(max) provides warning of possible toxic drug levels.

T_(max) was calculated and presented as median±range.

Pharmacokinetic Variables

For each subject, the following pharmacokinetic parameters werecalculated, whenever possible, based on the serum concentrations ofvitamin B₁₂ from Treatments A, B, and C, according to the modelindependent approach (Ref 1):

C_(max) Maximum observed concentration.

t_(max) Time to maximum concentration.

AUC_(0-t) Area under the concentration-time curve from time 0 to thetime of last measurable concentration, calculated by the lineartrapezoidal rule.

Pharmacokinetic calculations were performed, using SAS (SAS Inst.,Version 8.02).

Statistical Methods Planned in the Protocol and Determination of SampleSize

Statistical and Analytical Plans

Pharmacokinetic Analysis

Levels of vitamin B₁₂ in serum samples were measured as pg/mL. Serumconcentration values below the quantifiable limits of detection weretreated as zero. Actual sampling times, rather than scheduled samplingtimes, were used in all computations of the pharmacokinetic parameters.For ease of presentation, however, scheduled sampling times were used topresent results in tables, listings, and figures.

From the concentration data, non-compartmental pharmacokineticparameters (AUC_(0-t), C_(max), T_(max) were calculated as described inSection 8.4.3.

Statistical Analysis

All statistical tests were conducted at the 0.05 significance level,unless otherwise specifically identified. Summary statistics ofcontinuous parameters consisted of number (N), mean, median, SD, andrange.

Descriptive statistics were obtained and tabulated by treatment forlevels of vitamin B₁₂ at each time point and for the pharmacokineticparameters calculated.

Bioequivalence was evaluated for the test (Treatment A—Nasal Spray)versus the reference (Treatment B-Gel). An analysis of variance (ANOVA,Ref. 2) was performed and the 90% confidence intervals were generatedfor the ratio of test/reference. C_(max) and AUC_(0-t), were naturallog(log_(e)) transformed prior to analysis. The corresponding 90%confidence intervals for the geometric mean ratio were obtained bytaking the antilog of the 90% confidence intervals for the differencebetween the means on the log scale.

It was assumed that the test (Treatment A) is non-inferior (with respectto the reference (Treatment B) if the lower bound of the 90% confidenceintervals from log_(e)-transformed C_(max), and AUC_(0-t) were greaterthan or equal to 80%. If the lower bound of the 90% confidence intervalsfrom log_(e)-transformed C_(max) and AUC_(0-t) were less than 80%, itwas assumed that non-inferiority could not be established.

The sequence effect was tested using the mean square error (MSE) forsubject within sequence as the error term. All other main effects weretested against the MSE from the ANOVA model.

Bioavailability was evaluated for the test (Treatments A and B—NasalSpray and Gel, respectively) and the reference (Treatment C—IM) groups.Relative bioavailability was assessed by examining the 90% confidenceintervals for the ratio of the test (Treatments A and B) group meansrelative to the reference (Treatment C) group mean.

For T_(max), the analyses were run using Wilcoxon's matched pairs methodto determine if differences exist between the test group and eachreference group.

SUMMARY - CONCLUSIONS PHARMACOKINETIC RESULTS: The relativebioavailability for the two IN formulations was 0.9715. Bioavailabilitywhen comparing Treatment A (spray) versus Treatment C (IM) was 0.6105,and 0.6284 when comparing Treatment B (gel) versus Treatment C (IM). Thepharmacokinetic profiles of the spray formulation and the gelformulation were similar for C_(max) (1480 pg/mL, 1670 pg/mL,respectively) and AUC_(0-t) (92000 pg * hr/mL, 97000 pg * hr/mL,respectively). Additionally, the median difference for T_(max) betweenthe spray and gel IN formulation was less than 15 minutes (−0.24). TheC_(max) value for the IM formulation was significantly higher than theC_(max) values for the two IN formulations (p < 0.0001). Bioequivalencewas established for the Vitamin B₁₂ IN spray with regard to the gel databased on C_(max) and AUC_(0-t). The 90% confidence intervals for thelog_(e)-transformed C_(max) and AUC_(0-t) for the spray and gelformulations fell within the range of 80% to 125%. Additionally,non-inferiority can be assumed when comparing the two IN formulationsbecause the lower bounds of the confidence intervals are greater than80% for both AUC_(0-t) and C_(max). CONCLUSIONS: The relativebioavailability for the two IN formulations was 0.9715. Bioavailabilityfor Treatment A (spray) versus Treatment C (IM) was 0.6105, and 0.6284when comparing Treatment B (gel) versus Treatment C (IM). Thepharmacokinetic profiles of the spray formulation and the gelformulation are similar for C_(max) (1480 pg/mL, 1670 pg/mL,respectively) and AUC_(0-t) (92000 pg * hr/mL, 97000 pg * hr/mL,respectively). Additionally, the median difference for T_(max) betweenthe spray and gel IN formulation was less than 15 minutes (−0.24). TheC_(max) value for the IM formulation was significantly higher than theC_(max) values for the two IN formulations (p < 0.0001). Bioequivalencebetween the Vitamin B₁₂ spray formulation and the Vitamin B₁₂ gelformulation was established using log_(e)-transformed 90% confidenceintervals for AUC_(0-t) and C_(max). The 90% confidence intervals forthe log_(e)-transformed C_(max) and AUC_(0-t) for the spray and gelformulations fell within the range of 0.80 to 1.25. Noninferiority canbe assumed for the two IN formulations (Treatment A versus Treatment B).All Vitamin B₁₂ formulations were safe and well tolerated by healthymale and female volunteers.

EXAMPLE 2

We conducted a non-blinded, single dose, parallel group study to comparethe uptake of Vitamin B₁₂ into the cerebrospinal fluid (CSF) afterintranasal and intramuscular administration in healthy male andnon-pregnant female volunteers. This study compared CSF levels to plasmalevels produced by both formulations.

Thirty-six healthy male and non-pregnant female subjects, age 18 andover, were enrolled in the study. Eighteen subjects received a singleintranasal dose of 500 mcg delivered as a 0.1 mL spray and eighteensubjects received a single intramuscular dose of 100 mcg deliveredintramuscularly. Each subject visited the clinical site three times in aone-month period. These visits consisted of a screening visit, onedosing visit and a final visit.

After each dosing, each subject underwent lumbar puncture only once,with the retrieval of a total 4.0 mL of CSF (4 tubes, 1.0 mL per tube).One third of the subjects had a CSF sample collected at 60 minutes postdosing, one third of subjects had a CSF sample collected at 90 minutespost dosing, and one third of subjects had a CSF sample collected at 120minutes post dosing.

In addition to the above, on the day of dosing 7 mL blood samples weredrawn before dosing and post dosing at 5, 10, 15 and 20 minutes, and at0.5, 1, 1½, 2, 3, 4, 6, and 8 hours post-dose (prior to discharge).

The cerebrospinal fluid was evaluated for total Vitamin B₁₂ content. Itwas the objective of the study described herein to measure the amount ofVitamin B₁₂ present in the blood and CSF following intramuscular (IM)and nasal administration.

Reference and Test Products

Reference Product: Cyanocobalamin 100 mcg intramuscular injection.

Cyanocobalamin Injection, USP is a sterile solution of cyanocobalamin(Vitamin B₁₂) for intramuscular or subcutaneous injection. Each mLcontains 1,000 mcg cyanocobalamin.

Test Product: Vitamin B₁₂ Nasal Spray=500 mcg/0.1 mL spray. Thecyanocobalamin intranasal aqueous solution was a preferred formulationof the present invention and contained cyanocobalamin at a concentrationof 0.5% (percent of total weight), citric acid 0.12%, sodium citrate0.32%, glycerin 2.23%, 50% benzalkonium chloride solution 0.04% and96.79% water.

Vitamin B₁₂ Nasal Spray is supplied as a 2.3 mL bottle to deliver onedose: 500 mcg/0.1 mL per dose.

Before intranasal dosing, all subjects were be given an orientation ofthe proper dosing technique and general conduct of the study.

The subject was instructed to gently blow his/her nose. The subjectremained in a seated position, and the primed IN applicator was insertedinto the nostril by the subject, under the direction of the study staff.During dosing, the contralateral nostril was closed with the forefinger.Subjects will also be instructed to tilt their heads slightly back fordosing and to return their heads to an upright position while sniffingin gently immediately following dosing. A 0.1 mL dose of vitamin B₁₂spray will be released into the nasal cavity. (A dose is a singleapplication to one nostril.) Subjects must inform the staff if theysneeze or if the product drips out of their nose. Subjects will not bere-dosed if they sneeze or if the product drips out of their nose.Subjects will be instructed to refrain from blowing their nose for 1hour following IN treatment.

After dosing, each subject underwent lumbar puncture, involving theretrieval of 4.0 mL of CSF (4 tubes, 1.0 mL per tube). One third ofsubjects from each group will have a CSF sample collected at 60 minutespost dosing, one third of subjects will have a CSF sample collected at90 minutes post dosing, and one third of subjects will have a CSF samplecollected at 120 minutes post dosing.

At the appropriate time after dosing, the Investigator positioned thepatient appropriately in order to proceed with lumbar puncture. Thelumbar area was prepared and draped in the usual aseptic fashion. Localanesthesia was utilized (1% xylocaine, 1-5 mL). Upon reaching a state ofadequate anesthesia, a spinal needle (20 or 22 G) was introduced intothe spinal canal, at the level deemed appropriate by the Investigator.The CSF samples were collected 60, 90 or 120 minutes afteradministration. A total of 4.0 mL of CSF were collected from eachpatient, and distributed into 4 separate collection tubes. The tubeswere appropriately labeled with a patient identifier and submitted forbioanalytical analysis. Upon completion of CSF collection, the spinalneedle was removed.

The levels of vitamin B12 were determined in both the CSF and bloodserum using Vitamin B₁₂ concentrations in the CSF will be analyzed forQualitative determination of Vitamin B₁₂ using a validated TOSOH Nex. 1Aprocedure.

Results and Conclusion

The data showed that the ration of vitamin B₁₂ to serum was higher inthose individuals receiving intranasal administration of vitamin B₁₂than those receiving intramuscular injections of vitamin B₁₂.

The average ratio (B₁₂ CSF/B₁₂ Serum×100) ranged from 1.1 to 1.9 forthose individuals receiving intranasal administration of vitamin B₁₂while those who received intramuscular injections of vitamin B12 had anaverage ratio ranging from 0.17 to 0.24. This is a surprising result inthat intranasal administration only has about a 7-12% bioavailability inthe blood serum relative to intramuscular injection of vitamin B₁₂. Thisindicates that intranasal administration of vitamin B₁₂ reaches the CSFmuch more effectively than by intramuscular injection.

EXAMPLE 3 Production of a Cyanocobalamin Solution

A 4000 g batch of a cyanocobalamin solution of the present invention,which had a concentration of 500 mcg/0.1 g of solution.

Starting Materials I. Formula Record Ingredient Name Theoretical Weight(Grams) Cyanocobalamin, USP 20.0 Citric Acid, USP (Anhydrous) 4.8 SodiumCitrate, USP (Dihydrate) 12.8 Glycerin, USP 89.2 Benzalkonium ChlorideSolution, NF (50%) 1.6 Purified Water, USP 3871.6*

The 3871.6 grams of water was placed in a stainless steel container,which had been placed on a hot plate. The water was heated to about 30°C. and stirred. Into the heated water was added 12.8 g of sodium citratewhile the water was being stirred at 300 rpm for 5 minutes. The 4.8 g ofcitric acid was then added and stirred for 10 minutes. Into this mixturewas added 20.0 g of cyanocobalamin and stirred for 30 minutes at 30° C.at 300 rpm. The hot plate was then turned off. The 89.2 g of glycerinwas added and stirred for 5 minutes at 300 rpm. Into the cyanocobalaminsolution was then added 1.6 g of an aqueous solution containing 50% byweight of Benzalkonium Chloride was added to the solution and stirredfor 5 minutes at 300 rpm. The pH was then measured and adjusted if thepH was not with the 4.5-5.5 range. Additional water was added to bringthe weight of the solution to 4000 g.

EXAMPLE 4

This example describes a pharmaceutical composition product comprisingan aqueous solution formulation of salmon cyanocobalamin at aconcentration sufficient to produce therapeutically effective plasmaconcentrations and an actuator to produce an aerosol of said solution,wherein the spray pattern ellipticity ratio of said aerosol is between1.00 and 1.40 when measured at a height of 30 cm distance from theactuator tip.

The volume of the aerosol can be between about 5 microliters and 1.0 ml,preferably between 20 and 200 microliters.

This test method describes the procedure for characterizing plumegeometry of the cyanocobalamin nasal solution formulations using theSprayView NSP system. The plume geometry is characterized using aSprayView High Speed Optical Spray Characterization System (SprayViewNSP) with Integrated SprayView NSx actuation station (Image ThermEngineering, Inc., Sudbury, Mass.) according to the methods described inU.S. Pat. No. 6,665,421 and U.S. patent Application Publication No.20030018416 published Jan. 23, 2003.

Using the formulation of table 1 the spray characterization and dropletsize of the formulation in both a 3 mL bottle both having a nasal SprayPump w/Safety Clip, Pfeiffer SAP # 65550, which delivers a dose of 0.1mL per squirt and has a diptube length of 36.05 mm.

The droplet size data are shown in the following table.

Droplet Size for Nasal Spray Bottle and Pfeiffer SAP # 60548 % <10 D₁₀D₅₀ D₉₀ Span micrometer 1 mL Salmon 12.5 26.9 55.3 1.6 5.1cyanocobalaminBelow are listed the spray pattern results

Spray Pattern Major Axis Minor Axis Ellipticity Ratio active 3 mL 35.3mm 30.8 mm 1.14

1. A method for administering cyanocobalamin intranasally comprised ofproviding an aqueous solution of cyanocobalamin, wherein the solution ofcyanocobalamin has a viscosity of less than 1000 cPs, with the provisothat mercury and mercury containing compounds are not present in thesolution, wherein the cyanocobalamin formulation is administered into anose of an individual through an actuator tip as a spray, wherein thespray has a spray pattern ellipticity ratio of from about 1.0 to about1.4 when measured at a height of 3.0 cm from the actuator tip.
 2. Themethod of claim 1 wherein the spray produces droplets, wherein less than5% of the droplets are less than 10 μm in size.
 3. The method of claim 1wherein the spray has a spray pattern major axis and minor axis ofbetween 25 and 40 mm each.
 4. The method of claim 1 wherein the solutionof cyanocobalamin is further comprised of citric acid, and sodiumcitrate wherein the solution has a pH of from about 4-6.
 5. The methodof claim 4 wherein the pH of the solution is about
 5. 6. The method ofclaim 1 wherein cyanocobalamin is present in solution at a concentrationof between 0.5-11% by weight.
 7. The method of claim 6 wherein theconcentration of cyanocobalamin in solution is about 0.5%.
 8. The methodof claim 1 wherein the citric acid is present in solution at aconcentration of about 0.12%, and the sodium citrate is present insolution at a concentration of about 0.32%, in water.
 9. The method ofclaim 1 wherein the cyanocobalamin spray produces droplets of thesolution, and wherein 50% of the droplets are 26.9 μm or less in size.10. The method of claim 1 wherein the cyanocobalamin spray producesdroplets of the solution, and wherein 90% of the droplets are 55.3 μm orless in size.
 11. The method of claim 1 wherein the cyanocobalamin sprayproduces droplets of the solution, and wherein 10% of the droplets are12.5 μm or less in size.
 12. A method for administering cyanocobalamincomprised of providing an aqueous solution of cyanocobalamin whereinsaid aqueous solution of cyanocobalamin is comprised of cyanocobalaminat a concentration of about 0.5% of total weight of solution, citricacid at a concentration of about 0.12%, sodium citrate at aconcentration of about 0.32%, glycerin at a concentration of about2.23%, benzalkonium chloride at concentration of about 0.02% and water,wherein said solution of cyanocobalamin is suitable for intranasaladministration and has a viscosity less than about 1000 cPs, with theproviso that the solution of cyanocobalamin contains no mercury ormercury-containing compounds, and wherein the cyanocobalamin solution isadministered into a nose of an individual through an actuator tip as aspray, wherein the spray has a spray pattern ellipticity ratio of fromabout 1.0 to about 1.4 when measured at a height of 3.0 cm from theactuator tip.
 13. The method of claim 12 wherein the cyanocobalaminspray produces droplets of the solution, wherein less than 5% of thedroplets are less than 10 μm in size.
 14. The method of claim 12 whereinthe cyanocobalamin spray produces droplets of the solution, and wherein50% of the droplets are 26.9 μm or less in size.
 15. The method of claim12 wherein the cyanocobalamin spray produces droplets of the solution,and wherein 90% of the droplets are 55.3 μm or less in size.
 16. Themethod of claim 12 wherein the cyanocobalamin spray produces droplets ofthe solution, and wherein 10% of the droplets are 12.5 μm or less insize.
 17. The method of claim 12 wherein the spray has a spray patternmajor axis and a minor axis of about 25-40 mm each.
 18. A method forelevating the vitamin B12 levels in the cerebral spinal fluid (CSF)comprising administering intranasally a sufficient amount of a solutionof cyanocobalamin so that the average ratio of vitamin B12 in the CSF tothat in the blood serum (B12 CSF/B12 Serum×100) is increased to at leastabout 1.1, wherein said aqueous solution of cyanocobalamin is comprisedof cyanocobalamin at a concentration of about 0.5% of total weight ofsolution, citric acid at a concentration of about 0.12%, sodium citrateat a concentration of about 0.32%, glycerin at a concentration of about2.23%, benzalkonium chloride at concentration of about 0.02% and water,wherein said solution of cyanocobalamin is suitable for intranasaladministration and has a viscosity less than about 1000 cPs, with theproviso that the cyanocobalamin solution contains no mercury ormercury-containing compounds and wherein the cyanocobalamin solution isadministered into a nose of an individual through an actuator tip as aspray, wherein the spray has a spray pattern ellipticity ratio of fromabout 1.0 to about 1.4 when measured at a height of 3.0 cm from theactuator tip.
 19. The method of claim 18 wherein the cyanocobalaminspray produces droplets of the solution, wherein less than 5% of thedroplets are less than 10 μm in size.
 20. The method of claim 18 whereinthe cyanocobalamin spray produces droplets of the solution, and wherein50% of the droplets are 26.9 μm or less in size.
 21. The method of claim18 wherein the cyanocobalamin spray produces droplets of the solution,and wherein 90% of the droplets are 55.3 μm or less in size.
 22. Themethod of claim 18 wherein the cyanocobalamin spray produces droplets ofthe solution, and wherein 10% of the droplets are 12.5 μm or less insize.
 23. The method of claim 18 wherein the spray has a spray patternmajor axis and a minor axis of between 25-40 mm each.